Heat stress is a major environmental constraint for crop production worldwide. To respond to and cope with heat stress, plants synthesize heat shock proteins (HSPs), which are often molecular ...chaperones and are under the control of heat stress transcription factors (HSFs). Very little is known about the upstream regulators of HSFs. In a forward genetic screen for regulators of C-REPEAT BINDING FACTOR (CBF) gene expression (RCFs), we identified RCF2 and found that it is allelic to CPL1/FIERY2, which encodes a homolog of C-terminal domain phosphatase. Our results also showed that, in addition to being critical for cold stress tolerance, RCF2 is required for heat stress-responsive gene regulation and thermotolerance, because, compared with the wild type, the rcf2-1 mutant is hypersensitive to heat stress and because the reduced thermotolerance is correlated with lower expression of most of the 21 HSFs and some of the HSPs in the mutant plants. We found that RCF2 interacts with the NAC transcription factor NAC019 and that RCF2 dephosphorylates NAC019 in vivo. The nac019 mutant is more sensitive to heat stress than the wild type, and chromatin immunoprecipitation followed by quantitative PCR analysis revealed that NAC019 binds to the promoters of HSFA1b, HSFA6b, HSFA7a, and HSFC1. Overexpression of RCF2 or NAC019 in Arabidopsis thaliana increases thermotolerance. Together, our results suggest that, through dephosphorylation of NAC019, RCF2 is an integrator of high-temperature signal transduction and a mechanism for HSF and HSP activation.
Apple (Malus × domestica) trees are vulnerable to freezing temperatures. However, there has been only limited success in developing cold-hardy cultivars. This lack of progress is due at least partly ...to lack of understanding of the molecular mechanisms of freezing tolerance in apple.
In this study, we evaluated the potential roles for two R2R3 MYB transcription factors (TFs), MYB88 and the paralogous FLP (MYB124), in cold stress in apple and Arabidopsis. We found that MYB88 and MYB124 positively regulate freezing tolerance and cold-responsive gene expression in both apple and Arabidopsis.
Chromatin-Immunoprecipitation-qPCR and electrophoretic mobility shift assays showed that MdMYB88/MdMYB124 act as direct regulators of the COLD SHOCK DOMAIN PROTEIN 3 (MdCSP3) and CIRCADIAN CLOCK ASSOCIATED 1 (MdCCA1) genes. Dual luciferase reporter assay indicated that MdCCA1 but not MdCSP3 activated the expression of MdCBF3 under cold stress. Moreover, MdMYB88 and MdMYB124 promoted anthocyanin accumulation and H2O2 detoxification in response to cold.
Taken together, our results suggest that MdMYB88 and MdMYB124 positively regulate cold hardiness and cold-responsive gene expression under cold stress by C-REPEAT BINDING FACTOR (CBF)-dependent and CBF-independent pathways.
The apple is a favorite fruit for human diet and is one of the most important commercial fruit crops around the world. Investigating metabolic variations during fruit development can provide a better ...understanding on the formation of fruit quality. The present study applied a widely targeted LC-MS-based metabolomics approach with large-scale detection, identification and quantification to investigate the widespread metabolic changes during "Pinova" apple development and ripening. A total of 462 primary and secondary metabolites were simultaneously detected, and their changes along with the four fruit-development stages were further investigated. The results indicated that most of the sugars presented increasing accumulation levels while organic acid, including Tricarboxylic acid cycle (TCA) intermediates, showed a distinct decreasing trend across the four fruit-development stages. A total of 207 secondary metabolites consisted of 104 flavonoids and 103 other secondary metabolites. Many flavonoids maintained relatively high levels in the early fruit stage and then rapidly decreased their levels at the following developmental stages. Further correlation analyses of each metabolite-metabolite pair highlighted the cross talk between the primary and secondary metabolisms across fruit development and ripening, indicating the significant negative correlations between sugars and secondary metabolites. Moreover, transcriptome analysis provided the molecular basis for metabolic variations during fruit development. The results showed that most differentially expressed genes (DEGs) involved in the TCA cycle were upregulated from the early fruit stage to the preripening stage. The extensive downregulation of controlling genes involved in the flavonoid pathway is probably responsible for the rapid decrease of flavonoid content at the early fruit stage. These data provide a global view of the apple metabolome and a comprehensive analysis on metabolomic variations during fruit development, providing a broader and better understanding on the molecular and metabolic basis of important fruit quality traits in commercial apples.
Abstract
MdMYB88 and MdMYB124 have been demonstrated to be responsible for lignin accumulation in apple under drought stress. In this study, using a metabolomic approach, we identified differentially ...accumulated phenylpropanoid and flavonoid metabolites in
MdMYB88/124
transgenic RNAi plants under control and long-term drought stress conditions in apple roots. We confirmed the regulation of phenylalanine by MdMYB88 and MdMYB124 via UPLC-MS in apple roots under both control and drought conditions. Using Electrophoretic Mobility Shift Assay (EMSA) and ChIP-quantitative PCR (qPCR) analyses, we found that MdMYB88 positively regulates the MdCM2 gene, which is responsible for phenylalanine biosynthesis, through binding to its promoter region. Under long-term drought conditions,
MdMYB88/124
RNAi plants consistently accumulated increased amounts of H
2
O
2
and MDA, while
MdMYB88
and
MdMYB124
overexpression plants accumulated decreased amounts of H
2
O
2
and MDA. We also examined the accumulation of metabolites in the phenylpropanoid biosynthesis pathway in the leaves of
MdMYB88
and
MdMYB124
transgenic apple plants after long-term drought stress. We found that metabolites responsible for plant defense, including phenylpropanoids and flavonoids, accumulated less in the RNAi plants but more in the overexpression plants under both control and drought conditions. We further demonstrated that
MdMYB88/124
RNAi plants were more sensitive to
Alternaria alternata
f. sp.
mali
and
Valsa mali
, two pathogens that currently severely threaten apple production. In contrast,
MdMYB88
and
MdMYB124
overexpression plants were more tolerant to these pathogens. The cumulative results of this study provided evidence for secondary metabolite regulation by MdMYB88 and MdMYB124, further explained the molecular roles of MdMYB88 and MdMYB124 in drought resistance, and provided information concerning molecular aspects of their roles in disease resistance.
Cold stress resulting from chilling and freezing temperatures substantially reduces crop production worldwide. To identify genes critical for cold tolerance in plants, we screened Arabidopsis ...thaliana mutants for deregulated expression of a firefly luciferase reporter gene under the control of the C-REPEAT BINDING FACTOR2 (CBF2) promoter (CBF2:LUC). A regulator of CBF gene expression1 (rcf1-1) mutant that is hypersensitive to cold stress was chosen for in-depth characterization. RCF1 encodes a cold-inducible DEAD (Asp-Glu-Als-Asp) box RNA helicase. Unlike a previously reported DEAD box RNA helicase (LOW EXPRESSION OF OSMOTICALLY RESPONSIVE GENES4 LOS4) that regulates mRNA export, RCF1 does not play a role in mRNA export. Instead, RCF1 functions to maintain proper splicing of pre-mRNAs; many cold-responsive genes are misspliced in rcf1-1 mutant plants under cold stress. Functional characterization of four genes (PSEUDO-RESPONSE REGULATOR5 PRR5, SHAGGY-LIKE SERINE/THREONINE KINASE12 SK12, MYB FAMILY TRANSCRIPTION FACTOR CIRCADIAN1 CIR1, and SPFH/PHB DOMAIN-CONTAINING MEMBRANE-ASSOCIATED PROTEIN SPFH) that are misspliced in rcf1-1 revealed that these genes are cold-inducible positive (CIR1 and SPFH) and negative (PRR5 and SK12) regulators of cold-responsive genes and cold tolerance. Together, our results suggest that the cold-inducible RNA helicase RCF1 is essential for pre-mRNA splicing and is important for cold-responsive gene regulation and cold tolerance in plants.
Salt stress is an important environmental factor that significantly limits crop productivity worldwide. Studies on responses of plants to salt stress in recent years have identified novel signaling ...pathways and have been at the forefront of plant stress biology and plant biology in general. Thus far, research on salt stress in plants has been focused on cytoplasmic signaling pathways. In this study, we discovered a nuclear calcium-sensing and signaling pathway that is critical for salt stress tolerance in the reference plant Arabidopsis. Through a forward genetic screen, we found a nuclear-localized calcium-binding protein, RSA1 (SHORT ROOT IN SALT MEDIUM 1), which is required for salt tolerance, and identified its interacting partner, RITF1, a bHLH transcription factor. We show that RSA1 and RITF1 regulate the transcription of several genes involved in the detoxification of reactive oxygen species generated by salt stress and that they also regulate the SOS1 gene that encodes a plasma membrane Na(+)/H(+) antiporter essential for salt tolerance. Together, our results suggest the existence of a novel nuclear calcium-sensing and -signaling pathway that is important for gene regulation and salt stress tolerance.
Silver sulfide clusters with size less than 2 nm are of much interest in photoelectric devices, such as photoconductive devices, infrared detectors and superconductors. The electronic structures of ...Ag(Ag
2
S)
n
+
(
n
= 1–9) clusters are investigated using the combined method of genetic algorithm (GA) and density functional theory (DFT). Results reveal that S atoms prefer to be at the tip of the structure, and Ag atoms tend to form S-Ag-S unit between two S atoms. With the increase of cluster size, the open structure evolves into closed centre-hollow structure. According to the stability analysis, Ag(Ag
2
S)
6
+
is a magic number cluster due to its high HOMO-LUMO gap, big average energy and high second-order energy difference. Ag-S bonds, S-Ag-S and triangular Ag
3
S
3
units play key roles in stabilizing Ag(Ag
2
S)
n
+
clusters. According to the electron affinity energy analysis, the clusters are easier to get electrons when
n
= 1, 3, 5 and 9. Based on the charge analysis of the studied clusters, the charges on Ag and S atoms are positive and negative, respectively, which indicates that the charge transfer from Ag to S occurs in cluster. Infrared and Raman spectra are dependent on the symmetries of the clusters. The molecular orbitals of Ag(Ag
2
S)
6
+
and Ag(Ag
2
S)
8
+
present superatomic properties. We hope that the clusters studied here could provide valuable data for future experimental and theoretical study of cationic silver sulfide clusters.
Graphical abstract
a
The second-order energy differences (Δ
2
E
).
b
Energy gaps (
E
g
).
c
Average binding energies (
E
b
) versus cluster size,
n
Drought resistance in plants is influenced by multiple signaling pathways that involve various transcription factors, many target genes, and multiple types of epigenetic modifications. Studies on ...epigenetic modifications of drought focus on DNA methylation and histone modifications, with fewer on chromatin remodeling. Changes in chromatin accessibility can play an important role in abiotic stress in plants by affecting RNA polymerase binding and various regulatory factors. However, the changes in chromatin accessibility during drought in apples are not well understood. In this study, the landscape of chromatin accessibility associated with the gene expression of apple (GL3) under drought conditions was analyzed by Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) and RNA-seq. Differential analysis between drought treatment and control identified 23,466 peaks of upregulated chromatin accessibility and 2447 peaks of downregulated accessibility. The drought-induced chromatin accessibility changed genes were mainly enriched in metabolism, stimulus, and binding pathways. By combining results from differential analysis of RNA-seq and ATAC-seq, we identified 240 genes with higher chromatin accessibility and increased gene expression under drought conditions that may play important functions in the drought response process. Among them, a total of nine transcription factor genes were identified, including ATHB7, HAT5, and WRKY26. These transcription factor genes are differentially expressed with different chromatin accessibility motif binding loci that may participate in apple response to drought by regulating downstream genes. Our study provides a reference for chromatin accessibility under drought stress in apples and the results will facilitate subsequent studies on chromatin remodelers and transcription factors.
The phytohormone abscisic acid (ABA) is important for growth, development and stress responses in plants. Recent research has identified ABA receptors and signalling components that regulate seed ...germination and stomatal closure. However, proteins that regulate ABA signalling remain poorly understood. Here we use a forward-genetic screen to identify rbm25-1 and rbm25-2, two Arabidopsis mutants with increased sensitivity to growth inhibition by ABA. Using RNA-seq, we found that RBM25 controls the splicing of many pre-mRNAs. The protein phosphatase 2C HAB1, a critical component in ABA signalling, shows a dramatic defect in pre-mRNA splicing in rbm25 mutants. Ectopic expression of a HAB1 complementary DNA derived from wild-type mRNAs partially suppresses the rbm25-2 mutant phenotype. We suggest that RNA splicing is of particular importance for plant response to ABA and that the splicing factor RBM25 has a critical role in this response.
Experimental design and schematic diagram of the workflow used in this study. Two sets of biological replicate samples were analyzed by iTRAQ, using the hpRP-nanoLC–MS/MS workflow for examining ...proteome changes in apple trees in response to long term moderate drought. Proteins were identified using Mascot software.
•Higher Pn and photosynthesis mainly contribute to higher WUE.•Calvin cycle improves Pn in treated cv. ‘Qinguan’.•Stabilized photosynthetic electron transfer prevents photoinhibition and ROS.
Water use efficiency is an important indicator for plant adaptation and resistance to drought conditions. We previously found that under moderate drought stress, the water use efficiency of cv. ‘Qinguan’ apple (Malus domestica Borkh.) (tolerant to drought) was enhanced, while that of cv. ‘Naganofuji No. 2’ was not enhanced. In this research, we also found that instantaneous water-use efficiency of cv. ‘Qinguan’ was higher than that of cv. ‘Naganofuji No. 2’, mainly because of its higher net photosynthesis rate. To dissect the potential mechanisms underlying this phenomenon, we performed a comparative iTRAQ-based proteomics analysis with leaves of drought-treated cv. ‘Qinguan’ and ‘Naganofuji No. 2’. We identified 4078 proteins, of which 594 were differentially abundant between drought and well-watered leaves. The majority of increased proteins were predicted to be involved in photosynthetic pathway in drought treated cv. ‘Qinguan’ leaves, indicating that regulation of photosynthesis plays an important role for higher water use efficiency under drought stress. Enzyme activity assays were performed to validate the proteomics data. Our results suggested that the main regulatory mechanisms for high water use efficiency of cv. ‘Qinguan’ under moderate drought stress included the maintaining of Calvin cycle function by increasing key enzymes, stabilization of photosynthetic electron transfer and keeping reactive oxygen species at normal level by regulation of photosynthetic electron transfer chain, photorespiration and reactive oxygen species scavenging capability, thus prevented photoinhibition, reduced reactive oxygen species production and enhanced net photosynthesis rate. In addition, the response of signal regulatory proteins and abiotic stress-responsive proteins to drought also helped plants to cope with such stress.
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